Project Summary/Abstract The goal of this project is the development of an innovative platform for discovery of small molecule chemokine inhibitors. Chemokines direct cell migration primarily by binding and activating a family of G protein-coupled receptors that are post- translationally modified by tyrosine sulfation. Sulfation is required for chemokine function, suggesting that receptor sulfotyrosines participate directly in specific binding of the chemokine ligand. We first revealed the molecular basis for sulfotyrosine recognition in the chemokine family by solving the NMR structure of a complex between the chemokine SDF1/CXCL12 and a sulfotyrosine-containing fragment of its receptor CXCR4. The N-terminal extracellular domain of CXCR4 contains three potential sulfation sites, and each sulfotyrosine occupies a unique cleft on the CXC12 surface. In the initial funding period we showed that sulfotyrosine recognition sites are essential for high affinity receptor binding and can be targeted for inhibition using small molecules directed at the chemokine ligand. We also discovered an unprecedented example of autoinhibition in the chemokine family by an intrinsically disordered region of CCL21. In this renewal application, we propose to identify small molecule ligands of the three main pro-metastatic chemokines. We will screen for chemical fragments that bind the sulfotyrosine-recognition sites of CXCL12, CCL19 and CCL21and optimize them as competitive inhibitors of receptor binding that block cancer cell migration (aim 1), develop a chemical inducer of dimerization that converts endogenous CXCL12 into an inhibitory biased agonist (aim 2), and screen for molecules that bind and stabilize the autoinhibited form of CCL21 (aim 3). Structure-based drug discovery guided by sulfotyrosine recognition is a conceptual advance that can be applied to most of the 50 members of the chemokine family and other complexes that require this protein modification to function in the extracellular space. New cancer treatments that emerge from application of this strategy will have a major impact on human health.